Mechanisms of luminal protein targeting in kidney and hepatic epithelial cells Columnar epithelia (e.g. kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. They differ morphologically in that columnar epithelia establish their apical domain (AP) and basolateral domains (BL) at opposing poles whereas hepatocytes establish their AP domain, the bile caliculi (BC) in the midst of their BL domains. They also differ drastically in how they establish and maintain their surface domains. Whereas columnar epithelia target their plasma membrane (PM) proteins predominantly from the trans Golgi network (TGN) to the AP domain, hepatocytes target both AP and BL PM proteins from the TGN to the BL surface, from where AP proteins are sorted to BC by transcytosis. Although important progress has been made in understanding the trafficking routes, sorting compartments, and targeting mechanisms of columnar cells, particularly the kidney epithelial cell line MDCK, the trafficking routes, and sorting compartments and mechanisms of hepatocytes remain poorly understood. This is in large part due to the fact that hepatic cell lines are not amenable to trafficking studies with the classical biotin based targeting assays utilized in MDCK cells and are distinctly more difficult to transfect. We propose innovative experimental approaches to identify the trafficking itinerary, subcellular compartments and molecular machinery involved in the trafficking of model AP PM proteins in hepatic WIFB cells, comparatively with MDCK cells. Our hypotheses are: (i) novel imaging technologies and (ii) analysis of the post-exocytic fate of AP proteins provide enough resolution to understand the different sorting and trafficking strategies of hepatocytes and columnar cells and (iii) the serine/threonine kinase Par1b acts as a molecular switch between the direct and transcytotic pathways. We propose the following Specific Aims: Aim 1: To quantitatively assess the extent of vectorial and transcytotic delivery of model apical PM proteins in WIFB and MDCK cells. Aim 2: To define comparatively the site of exocytic sorting of AP and BL markers in WIFB and MDCK cells. Aim 3: To identify the machinery used by AP PM proteins to exit the TGN in WIFB and MDCK cells. With our novel approaches we will set the framework for a long overdue comparative molecular model of protein targeting in hepatic versus columnar cells. This is a question of high priority for epithelial polarity that has remained unanswered for more than 25 years. PUBLIC HEALTH RELEVANCE: Epithelia are tissues of tightly adherent cells that enclose all organs and thus separate and protect them from the surrounding milieu. Yet far from putting up a passive protective wall, epithelial layers act as sophisticated gatekeepers that mediate the controlled traffic of molecules such as gas, ions, nutrients and waste products in and out of the organs they surround, often against a concentration gradient. To fulfill this function, epithelia form polarized membrane domains that restrict the transporters and gated channels responsible for the trafficking of molecules across the cell membrane to either the "inside" or "outside" facing surfaces of the epithelia, thus ensuring the vectorial transport of molecules. Most epithelia, such as those of the kidney, intestinal tract, lungs or mammary gland organize in two cells layers to enclose a central lumen, forming the tubes, ascini or aveoli through which urine, nutrients, milk or air are transported. The surface domain facing the lumen is called apical or luminal surface. Liver epithelia, by contrast, organize in one-cell thick plates and carve out a lumen, the bile canalicular network, by interrupting the lateral domains of neighboring cells. Loss or disorganization of epithelial luminal sufaces is a feature of many cancers. It is also hallmark of other pathologies such as microvilli inclusion disease and chronic inflammation of the liver (cholestasis). Thus, understanding how luminal surfaces are formed and maintained is one of the central questions in epithelial cell biology. This proposal addresses one important aspect of lumen formation. That is: How do kidney and liver epithelial cells target membrane proteins specifically to their luminal domains? It is known that both cell types utilize radically different strategies for luminal proteins targeting, but the molecular mechanisms that distinguish them are not known. We have developed novel tools and approaches to elucidate key features of the luminal protein trafficking machinery characteristic of the two major epithelial phenotypes represented by the kidney and the liver.